Multiple Loci Associated with Renal Function in African Americans

<div><p>The incidence of chronic kidney disease varies by ethnic group in the USA, with African Americans displaying a two-fold higher rate than European Americans. One of the two defining variables underlying staging of chronic kidney disease is the glomerular filtration rate. Meta-analysis in individuals of European ancestry has identified 23 genetic loci associated with the estimated glomerular filtration rate (eGFR). We conducted a follow-up study of these 23 genetic loci using a population-based sample of 1,018 unrelated admixed African Americans. We included in our follow-up study two variants in <em>APOL1</em> associated with end-stage kidney disease discovered by admixture mapping in admixed African Americans. To address confounding due to admixture, we estimated local ancestry at each marker and global ancestry. We performed regression analysis stratified by local ancestry and combined the resulting regression estimates across ancestry strata using an inverse variance-weighted fixed effects model. We found that 11 of the 24 loci were significantly associated with eGFR in our sample. The effect size estimates were not significantly different between the subgroups of individuals with two copies of African ancestry <em>vs</em>. two copies of European ancestry for any of the 11 loci. In contrast, allele frequencies were significantly different at 10 of the 11 loci. Collectively, the 11 loci, including four secondary signals revealed by conditional analyses, explained 14.2% of the phenotypic variance in eGFR, in contrast to the 1.4% explained by the 24 loci in individuals of European ancestry. Our findings provide insight into the genetic basis of variation in renal function among admixed African Americans.</p> </div

Characteristics of the study sample.

a<p>Ranges are presented as median (first quartile, third quartile).</p

Association with eGFR stratified by ancestry.

a<p>Positions are based on NCBI build 36.</p>b<p>Shown are the nominal numbers of SNPs in the set for each locus.</p>c<p>“DoF” indicates the effective degrees of freedom for each locus, which is the correction factor used to adjust <i>p</i>-values.</p>d<p>β_meta and SE_meta refer to the estimates from the meta-analysis combined across the three strata of local ancestry.</p

A human tissue-based functional assay platform to evaluate the immune function impact of small molecule inhibitors that target the immune system

<div><p>While the immune system is essential for the maintenance of the homeostasis, health and survival of humans, aberrant immune responses can lead to chronic inflammatory and autoimmune disorders. Pharmacological modulation of drug targets in the immune system to ameliorate disease also carry a risk of immunosuppression that could lead to adverse outcomes. Therefore, it is important to understand the ‘immune fingerprint’ of novel therapeutics as they relate to current and, clinically used immunological therapies to better understand their potential therapeutic benefit as well as immunosuppressive ability that might lead to adverse events such as infection risks and cancer. Since the mechanistic investigation of pharmacological modulators in a drug discovery setting is largely compound- and mechanism-centric but not comprehensive in terms of immune system impact, we developed a human tissue based functional assay platform to evaluate the impact of pharmacological modulators on a range of innate and adaptive immune functions. Here, we demonstrate that it is possible to generate a qualitative and quantitative immune system impact of pharmacological modulators, which might help better understand and predict the benefit-risk profiles of these compounds in the treatment of immune disorders.</p></div

Immune function impact profiles of small molecule inhibitors.

<p>Small molecule inhibitors were evaluated in six different functional assays with nine different read-outs. For a given compound on the Y-axis, each symbol on the X-axis represents the protein binding corrected cellular potency of that compound in the corresponding assay. The potencies of the evaluated compounds in these assays are displayed as IC₅₀ values in the X-axis. The reported IC₅₀ values were generated from a composite of 8–10 point dose response curves from n = 6–8 donors for each compound in each assay. Immune function impact of a broader set (A) or a subset (B) of inhibitors are presented.</p

Assays to evaluate immune function impact of small molecule inhibitors.

<p>All the assays were established using healthy donor peripheral blood mononuclear cells (PBMCs) or whole blood. The immune functions evaluated, the tissues analyzed, stimulations used as well as the tissue analysis methods are listed for each assay. Small molecule inhibitors that are known to inhibit these functions were used as positive controls (assay controls) in each run of the assay, to ensure consistent technical performance and data robustness.</p

A conceptual framework to develop an ‘immune impact score’ for compounds.

<p>The impact of compounds in immune system-based functional assays, -omics approaches, PK/PD information and chemical informatics (that classify and predict compound-function relationships based on available information) might be integrated with the aim of assigning an ‘immune impact score’ for individual compounds. Further integration of available or modeled efficacy and adverse event data for compounds with the immune impact score might be useful in better understanding and predicting the efficacy, adverse event profile and differentiation of these compounds in a clinical setting. PK/PD–Pharmacokinetics/Pharmacodynamics, AE–adverse events.</p

Impact of SYK/ZAP-70 inhibitor and prednisolone on gene expression profiles in the T cell stimulation assay.

<p>(A) A nanostring gene expression panel was used to evaluate mRNA profiles of PBMCs 3, 6 and 24 h following treatment with three different concentrations of SYK/ZAP-70 inhibitor and prednisolone. The gene expression profiles of unstimulated and compound treated stimulated samples are shown. All data were normalized to housekeeping genes and stimulated DMSO control samples. Hierarchical agglomerative clustering of genes with greater than a 2-fold change (p-value<0.05) is shown. Transcript expression of IL-2 under unstimulated, stimulated, and stimulated plus SYK/ZAP-70 inhibitor or prednisolone treated conditions at the 3h (B), 6h (C) and 24h (D) time points in the T cell stimulation assay. The gene expression profiles in (A) is a composite of PBMCs from n = 3 donors, while the individual gene expression profiles in (B-D) are mean±SEM of mRNA expression from n = 3 PBMC donors.</p

The effect of small molecule inhibitors on reactive oxygen species (ROS) production by human phagocytes.

<p>Ten minutes following stimulation with opsonized bacteria, the production of ROS in neutrophils and monocytes from human whole blood was evaluated using flow cytometry. Small molecule inhibitors inhibit ROS production neutrophils (A) and monocytes (B) in human whole blood following stimulation with opsonized bacteria. For each compound, the potency (red circles) and the percentage maximal inhibition achieved in the assay (black squares) are plotted along the X-axis. The potencies of the evaluated compounds in these assays are displayed as IC₅₀ values. A maximum of 100% inhibition is possible for small molecule inhibitors in these assays. The reported potency and % max inhibition values were generated from a composite of 8–10 point dose response curves from n = 6–8 donors for each compound. The positive control used in this assay (Diphenyliodonium chloride [DPI], an NADPH oxidase inhibitor), can inhibit ROS production from neutrophils (C) or monocytes (D) in a dose dependent manner. A BTK inhibitor consistently inhibited ~50% neutrophil ROS production (E) across multiple donors evaluated in this assay. Each point in the dose response curve indicates mean±SEM of % inhibition at that dose from n = 6–8 donors.</p

The impact of small molecule inhibitors on human T cell function.

<p>The production of IL-2 as well as the proliferation of CD4⁺ and CD8⁺ T cells was evaluated in a human <i>in vitro</i> PBMC assay. The production of IL-2 was evaluated 24h following stimulation with α-CD3 and α-CD28 while the proliferation of CD4⁺ and CD8⁺ T cells was evaluated in the same cultures 72h following α-CD3 and α-CD28 stimulation. Small molecule inhibitors suppress α-CD3 and α-CD28 induced IL-2 production (A), CD4⁺ T cell proliferation (B) and CD8⁺ T cell proliferation (C) in human PBMCs. For each compound, the potency (red circles) is plotted along the top X-axis and the percentage maximal inhibition achieved in the assay (black squares) are plotted along the bottom X-axis. The potencies of the evaluated compounds in these assays are displayed as IC₅₀ ovalues. A maximum of 100% inhibition is possible for small molecule inhibitors in these assays. The reported potency and % max inhibition values were generated from a composite of 8–10 point dose response curves from n = 6–8 donors for each compound.</p